The present invention relates to analytical methods, kits, electrodes and compositions for analysis of analytes in fluid samples. More particularly, the present invention relates to methods, kits, electrodes and compositions for detection of an analyte of interest in a clinical sample, and principally to compositions, methods, kits and electrodes useful in the analysis of lithium in saliva and further in sera, plasma, urine, cerebro-spinal fluid, whole blood or other biological (clinical) fluids, utilizing potentiometric ion selective electrodes.
Lithium, usually in the form of lithium carbonate or other lithium salts, is commonly used for the treatment of manic, manic depressive, hyper active, violent patients, etc. Although traces of lithium ion are distributed widely throughout the body, the major effect of exogenous lithium is upon the central nervous system (CNS).
Unlike other antipsychotic pharmaceutical agents, lithium is not thought to possess any general sedative properties, and, in therapeutic amounts, CNS effects are not observed except during chronic administration of lithium in manic or manic-depressive patients.
The exact therapeutic mechanism of lithium is not currently known, primarily because the pathophysiology of manic disorders is unknown.
Lithium ions are readily absorbed when given orally, and a plasma lithium peak is reached approximately 2-4 hours after ingestion of lithium carbonate. Lithium plasma levels are usually monitored at least twice weekly in new patients in order to maintain the level thereof within the range of from about 0.2 to about 1.0 mmole per liter. For severe cases of mania, however, the lithium plasma level may be increased to about 1.5 mmole per liter.
Therapeutic doses of lithium can cause fatigue, muscular weakness, slurred speech, ataxia, tremor of the hands, nausea, vomiting, diarrhea and thirst. At plasma levels above about 2.2 mmole per liter, more serious toxic effects occur, with the CNS primarily affected, i.e., consciousness is impaired, coma may occur, muscular rigidity, hyperactive deep reflexes and marked tremor and muscular fasciculations are observed, epileptic seizures can occur, and EEG abnormalities are common. A lithium plasma level of about 3.0 mmole per liter can be fatal.
Accordingly, it is not only important, but essential, to know what such plasma level is in order to avoid the deleterious effects associated therewith.
Analysis of lithium in biological fluids, such as sera, plasma, urine, cerebro-spinal fluid, or whole blood, is hampered by the presence of other ionic compounds in such fluids, and in particular, sodium ions. Analysis of lithium in saliva is particularly hampered by the presence of high calcium concentrations, which is present in much lower concentrations in the biological fluids listed above. These interferences are most noticeable at lower lithium concentrations (for example, about 0.10 mmole per liter).
With respect to serum of non-treated individuals, the molar ratio of lithium to sodium is about 1:1500. With respect to saliva, the molar ratio of lithium to calcium is about 1:1000. Accordingly, one of the challenges facing those developing an assay method for lithium is to overcome the interference from such ions.
Flame photometry is one method for lithium analysis in clinical specimens. In flame photometry, atoms are excited to an energy level above their ground state by a flame. Upon return to ground state, the energy is released as radiation at a frequency characteristic of the element under investigation. By measuring the emission light intensity, the concentration of the analyte of interest in the sample can be determined.
Despite its relative simplicity, flame photometry is a tedious procedure and includes several critical disadvantages as an analytical method, e.g., spectral interference between two or more substances in a sample (such as in the case with serum sodium and lithium or saliva calcium and lithium), background interferences, anionic and cationic interferences, and self-absorption.
Furthermore, routine maintenance of the instrumentation is not only critical to ensure good analytical results, but is itself a tedious procedure. Additionally, flame photometry instruments utilize air compressors, which on the whole are noisy, a distinct disadvantage in a clinical setting.
Finally, there is the practical concern of safely storing a flammable gas in a laboratory environment.
Thus, currently, the monitoring of lithium concentration within the blood involves a lengthy procedure which requires the employment of expensive non-safe equipment and professional operators for operating such equipment. A blood sample is extracted from the patient by a family doctor or a nurse. The extracted blood is collected within a sample tube. Following the addition of an anti-clotting material, the sample tube is transferred to a laboratory for analysis. At the laboratory, the plasma of the blood sample is separated from the blood cells by means of a centrifuge. The plasma is then transferred to another tube and is then analyzed by means of a flame photometer. The flame photometer is an expansive and cumbersome device which requires a professional operator. Further, the flame photometer is expensive to operate and has to be specifically prepared and calibrated prior to each examination. As a result, such device is usually activated only once or twice a week, after a substantial amount of blood samples have been collected. Following analysis of the blood sample, a written result is sent to the office and then to the family doctor. Such lengthy process (which may be extended for more than one week) may be ineffective in the sense that it does not provide an immediate feedback in the event of toxicity. Toxic levels of lithium within the patient's blood may occur as a result of introduction of over-dosage of the drug, or in the event of impaired clearance of the drug owing to damaged kidney or damaged liver. Lithium concentration within the blood is also dependent on hormonal regulation and other physiological factors and therefore may feature abnormal profile when the patient suffers from hormonal or other physiological problems. An immediate feedback is extremely important when treating children since relatively low concentrations of lithium may cause toxicity. Since there is no current effective treatment for lithium poisoning, an immediate feedback relating to lithium concentration within the blood is essential.
Ion selective electrode (ISE) technology is an alternative to flame photometry which avoids many of these problems. ISE technology involves the use of a reference electrode and an ion selective electrode separated by a membrane. The ion selective electrode is specific or sensitive to the particular ion of interest. Typically, the reference electrode and the ion selective electrode are simultaneously immersed into a sample solution. An electrical potential is developed between the electrodes which is relative to the presence of the ion to which the ISE is sensitive or specific. This potential can be utilized to determine the concentration of that ion in the sample. Most often the investigator desires to only measure the concentration of one ion out of the many different ions in solution. Thus, the ion selective composition of the ISE, referred to as a "carrier" or "ionophore", must be capable of sequentially complexing the desired ion, transporting the complexed ion across the membrane, and releasing the ion in preference to all other ions which may be present in the sample solution.
Macrocyclic polyethers, also referred to as cryptanols or "crown ethers", are well known lithium ion-complexing compounds which are suitable for use as ion selective electrodes. Such ionophores are described in, for example, U.S. Pat. Nos. 4,214,968; 4,504,368; Oesch, U. et al., "Ion Selective Membrane Electrodes for Clinical Use", Clin. Chem. 32:1448-1459 (1996); Kitayama, S. et al. "Lithium-Selective Polymeric Electrodes Based on Dodecylmethyl-14-Crown-4", Analyst 110:295-299 (1985); and, Attiyat, A. et al., "Comparative Evaluation of Neutral and Proton-Ionizable Crown Ether Compounds as Lithium in Ion-Selective Electrodes and in Solvent Extraction", Anal. Chem. 60:2561-2564 (1988). Other lithium ion complexing compounds are also available, and examples of these are described in U.S. Pat. No. 4,853,090; Gadzekpo, V. P. Y. et al., "Lipophilic Lithium Ion Carrier in a Lithium Ion Selective Electrode", Anal. Chem. 57:493-495 (1985); and, Gadzekpo, V. P. Y. et al., "Problems in the Application of Ion-Selective Electrodes to Serum Lithium Analysis", Analyst, 111:567-570 (1986). All of the preceding references of this paragraph are incorporated herein by reference as if fully set forth herein. Carriers or ionophores specific to ions other than lithium and to a variety of drugs are also well known in the art. Examples include, but are not limited to, calcium ionophore III (A23187 calcimicine). All of the preceding references of this paragraph are incorporated herein by reference as if fully set forth herein.
Two methods are associated with ISE: the direct potentiometric method, where the sample is measured directly; and the indirect potentiometric method, where the sample is diluted prior to analysis. Of the two, the indirect potentiometric method is in some aspects preferred because: (a) dilution of the sample derives the advantages of mass action law, (b) serum results do not usually correlate well between flame photometry and the direct potentiometric method, and (c) maintenance of a direct ISE analyzer is more difficult than an indirect ISE due to protein build-up on the direct ISE.
However, despite the advantages of the indirect potentiometric methodology, there are no indirect potentiometric analyzers for lithium commercially available. This is based, in part, on the relationship of lithium in a clinical specimen to other ions therein. For example, in serum, where as previously noted the ratio of lithium to sodium is 1:1500, or in saliva, where as previously noted the ratio of lithium to calcium is 1:1000. Furthermore, dilution of a sample makes analysis of the already miniscule amount of lithium present very difficult.
U.S. Pat. Nos. 5,110,742 and 5,288,678 teach indirect potentiometric method and diluent for analysis of lithium. The diluent includes effective amounts of a pH buffer and a non-cationic surfactant comprising at least one hydrophobic group, at least one hydrophilic group and being substantially free of polyoxyethylene groups. The pH buffer is preferably tris-(hydroxymethyl) aminomethane-phosphate, and the surfactant is 2,4,9,7-tetramethyl-5-decyn-4,7 diol. An indirect potentiometric determination of lithium in a clinical sample comprises according to U.S. Pat. Nos. 5,110,742 and 5,288,678 the steps of mixing the sample with a diluent, contacting an aliquot of the diluted sample with a lithium specific ion selective electrode and at least one ion selective electrode specifically responsive to a monovalent interfering ion, and measuring both the response of the lithium specific ion selective electrode and the monovalent interfering ion specific ion selective electrode, the responses being an indication of the concentration of lithium in the sample.
This method suffers a disadvantage, because two or more ions are to be measured instead of a single ion of interest. This calls for a plurality of sample electrodes being employed, each has to be calibrated before use, etc. Furthermore, this method is not applicable for saliva lithium determinations because the major interfering ion in saliva is the divalent ion calcium.
Indeed, it was thus far never attempted nor was it suggested to specifically monitor lithium concentration is saliva using potentiometric methods, either direct or indirect. However, being completely non-invasive, there is a long felt need for potentiometric methods adapted for saliva lithium determinations.
It is well known that any ISE is characterized by a given range of concentrations in which the potential between the ISE and the reference electrode is proportional to the concentration. This range is typically referred to as the linear range. Measurements of concentrations outside the linear range are either non-informative because of unavailability of a connecting function or grossly inaccurate. This phenomenon is even intensified in the presence of interfering ions, especially if present at relative high concentrations. The prior art fails to teach effective strategies of measuring ion concentrations falling below the linear range of their respective ISE.
Thus, a need exists for a simple and reliable potentiometric method for the determination of lithium in clinical samples, especially in saliva.